Method For Selective Derivatization of Oligohistidine Sequence of Recombinant Proteins

ABSTRACT

Methods and compositions for the selective derivatization of a oligohistidine-tagged recombinant protein. A modifying compound comprised of an imidazole reactive group, a linker, and a ligating group is contacted with the recombinant protein, and a cooperative bond forms between the ligating group and the oligohistidine tag in the presence of a metal cation, and a covalent bond forms between the imidazole reactive group and an imidazole ring of the oligohistidine tag followed by the concomitant separation of the imidazole reactive group from the linker. Addition of a metal chelator results in the dissociation of the ligating group and the oligohistidine tag.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/680,821, filed on Aug. 8, 2012 and entitled “Method forSelective Derivatization of Oligohistidine Sequence of RecombinantProteins,” the entire disclosure of which is incorporated herein byreference.

BACKGROUND

The present invention relates to methods for modification of proteinsand, more specifically, to methods for the chemical derivatization ofproteins.

The most common method of preparation of proteins involves preparationof recombinant DNA plasmid encoding the desired amino acid sequence,introduction of the recombinant DNA into prokaryotic or eukaryoticcells, and expression of the encoded protein in the cells. To simplifysubsequent purification of these proteins recombinant DNA sequencescommonly incorporate a nucleotide sequence encoding an oligohistidinetag at the beginning or at the end of the polypeptide chain. Duringpurification of recombinant proteins the oligohistidine sequence isbound on chromatography columns functionalized by aminocarboxylategroups binding divalent metal cations resulting in its separation fromother proteins that does not contain oligohistidine sequences.

Chemical derivatization of native and recombinant proteins is used formodification of their physical, chemical, and biological properties, andfor their immobilization on interfaces. Existing methods of chemicalderivatization of proteins involve formation of covalent bonds withamino acid residues of the polypeptide chain of proteins using reactionsof alkylation, acylation, formation of thioureas, formation of mixeddisulfides, as well as other reactions. These reactions are frequentlynot selective and provide complex mixtures of products.

BRIEF SUMMARY

Systems and methods for selective chemical reaction with anoligohistidine tag in a recombinant protein resulting in selectivechemical modification of proteins. According to one aspect is a methodfor the selective derivatization of a recombinant protein comprising thesteps of: (i) contacting a recombinant protein comprising aoligohistidine tag with a modifying compound in the presence of a metalcation, wherein the modifying compound comprises, for example, theformula X₁X₂X₃ wherein X₁ is an imidazole reactive group, X₂ is alinking group, and X₃ is at least one ligating group; wherein theimidazole reactive group comprises a functional group, an electronwithdrawing group, and a connector connecting the imidazole reactivegroup to X₂; and further wherein the contacting is performed underconditions sufficient for the formation of a coordinative bond betweenX₃ and the oligohistidine tag utilizing the metal cation, and underconditions sufficient for the formation of a covalent bond between X₁and an imidazole ring of the oligohistidine tag and concomitantseparation of X₁ and X₂; and (ii) breaking the bond between X₃ and theoligohistidine tag using a metal chelator.

According to an aspect, there is provided a imidazole reactive grouprepresented by Formula (I) or (II) below:

where R is the functional group, R1 is the electron withdrawing group,and R2 is the linking group. According to one embodiment, X₃ comprises aplurality of ligating groups.

According to another aspect, the electron withdrawing group is selectedfrom the group consisting of an acyl group, an ester group, a carbamylgroup, an alkylsulfonyl group, an arylsulfonyl group, a nitro group, acyano group, and combinations thereof.

According to yet another aspect, the functional group is selected fromthe group consisting of a fluorophore, an azido group, an alkyne group,an alkene group, a diene group, a triene group, a biotin residue, aphenyl, a substituted phenyl, a polycyclic aromatic group, an aromaticheterocyclic group, a polycyclic aromatic heterocyclic group, aradioactive group, a group that possess distinct magnetic properties,and combinations thereof.

According to one embodiment, X₃ is selected from the group consisting ofa primary amino group, a secondary amino group, a teriary amino group, acarboxy group, an imino group, a phenol group, a catechol group, ahydroxamate group, a sulfamido group, a phosphoryl group, an imidazolylgroup, a thiazolyl group, an oxazolyl group, a pyridazolyl group, apyridyl group, a thiol group, and combinations thereof.

According to another aspect, the metal cation is selected from the groupconsisting of Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺, Co²⁺, Co³⁺, Fe³⁺, and combinationsthereof. According to one embodiment, the conditions sufficient for theformation of a coordinative bond between X₃ and the oligohistidine tagcomprise the presence of an additive, which can be, for example,imidazole, N-methylimidazole, or N-alkylimidazole.

According to an aspect is a compound for the selective derivatization ofa recombinant protein comprising a oligohistidine tag, the compoundcomprising the formula X₁X₂X₃ wherein X₁ is an imidazole reactive group,X₂ is a linking group, and X₃ is at least one ligating group, furtherwherein the imidazole reactive group comprises a functional group, anelectron withdrawing group, and a connector connecting the imidazolereactive group to X₂, and further wherein the compound is adapted toform a bond between X₃ and a oligohistidine tag of a recombinant proteinin the presence of a metal cation, and further adapted to form acovalent bond between X₁ and an imidazole ring of the oligohistidinetag.

According to one aspect the imidazole reactive group of the compound orthe selective derivatization of a recombinant protein comprising aoligohistidine tag comprises the structure represented by Formula (I)below:

where R is the functional group, R1 is the electron withdrawing group,and R2 is the linking group.

According to an aspect, the functional group is selected from the groupconsisting of an azido group, an alkyne group, an alkene group, a dienegroup, a triene group, a biotin residue, a phenyl, a substituted phenyl,a polycyclic aromatic group, an aromatic heterocyclic group, apolycyclic aromatic heterocyclic group, and combinations thereof.

According to one embodiment, X₃ is selected from the group consisting ofa primary amino group, a secondary amino group, a teriary amino group, acarboxy group, an imino group, a phenol group, a catechol group, ahydroxamate group, a sulfamido group, a phosphoryl group, an imidazolylgroup, a thiazolyl group, an oxazolyl group, a pyridazolyl group, apyridyl group, a thiol group, and combinations thereof.

According to an aspect is a method for selective derivatization of arecombinant protein, the method comprising the steps of: (i) contactinga recombinant protein comprising a oligohistidine tag with a modifyingcompound in the presence of a divalent metal cation, wherein themodifying compound comprises the formula X₁X₂X₃, wherein X₁ is animidazole reactive group comprising the structure represented by Formula(I) or (II) below:

wherein R is a functional group, R1 is an electron withdrawing groupselected from the group consisting of an acyl group, an ester group, analkylsulfonyl group, an arylsulfonyl group, a nitro group, a cyanogroup, and combinations thereof, and R2 is a linking group; X₂ is thelinking group; and X₃ is a plurality of ligating groups each selectedfrom the group consisting of a primary amino group, a secondary aminogroup, a teriary amino group, a carboxy group, a phosphoryl group, animidazolyl group, a thiazolyl group, an oxazolyl group, a pyridazolylgroup, a pyridyl group, and combinations thereof; and further whereinthe contacting is performed under conditions sufficient for theformation of a bond between X₃ and the oligohistidine tag utilizing ametal cation, and under conditions sufficient for the formation of acovalent bond between X₁ and an imidazole ring of the oligohistidine tagand concomitant separation of X₁ and X₂; and (ii) breaking the bondbetween X₃ and the oligohistidine tag using a metal chelator.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The present invention will be more fully understood and appreciated byreading the following Detailed Description in conjunction with theaccompanying drawings, in which:

FIG. 1 is a schematic representation of a reaction scheme for thesynthesis of a selective derivatization of an oligohistidine sequence ofa recombinant protein according to an embodiment;

FIG. 2 is a schematic representation of a reaction scheme for theselective derivatization of an oligohistidine sequence of a recombinantprotein according to an embodiment;

FIG. 3 is a schematic representation of a reaction scheme for theselective derivatization of an oligohistidine sequence of a recombinantprotein according to an embodiment;

FIG. 4 is a schematic representation of a reaction scheme for theselective derivatization of an oligohistidine sequence of a recombinantprotein according to an embodiment;

FIG. 5 is a schematic representation of a reaction scheme for thederivatization of a recombinant protein with a fluorescent groupaccording to an embodiment; and

FIG. 6 is a schematic representation of a reaction scheme according toone embodiment for the synthesis of an ester used in the derivatizationof a recombinant protein with a fluorescent group.

DETAILED DESCRIPTION

Referring now to the drawings, wherein like reference numerals refer tolike parts throughout, there is seen in FIG. 1 a schematicrepresentation of a reaction scheme for the synthesis of a selectivederivatization of an oligohistidine sequence of a recombinant proteinaccording to an embodiment. According to an aspect of the invention aremethods and systems for selective chemical reaction with anoligohistidine tag in a recombinant protein resulting in selectivechemical modification of that protein.

According to another aspect are novel chemical reagents possessing atleast two different functional groups. The first functional group,depicted as group “A” in FIG. 1, comprises one, two, three, or moreligating components depicted as “L:”. These ligating groups are adaptedto be capable of coordinative binding through a divalent metal cationM²⁺ to an oligohistidine tag present in a recombinant protein. Thesecond functional group, depicted as group “IRG” in FIG. 1, comprises anImidazole Reactive Group (“IRG”) which is adapted to be capable ofselective alkylation of an imidazole ring of a histidine residue of theoligohistidine tag through an addition-elimination process. According toan embodiment, these two functional groups (A and IRG) are connected bycovalent bonds to a linker as represented by, for example, structure 1in FIG. 1 and structure 5 in FIG. 2. Because the coordinative binding isspecific to an oligohistidine tag that is rarely present in polypeptidechains of proteins, the disclosed method allows selective derivatizationof the oligohistidine sequence in the presence of other amino acidresidues in the same recombinant protein, and in the presence of otherproteins, either naturally- or artificially-occurring.

In a first embodiment schematically represented in FIG. 1, functionalgroups A and IRG in structure 1 are connected through a carbon-oxygenbond that undergoes elimination on a subsequent step of theabovementioned process. The initial rapid and reversible coordinativebinding of ligating groups L: in new reagents through the metal cationM²⁺ to the oligohistidine tag produces mixed metal complex 2 shown inFIG. 1. The mixed metal complex 2 serves as a template for thesubsequent addition-elimination reaction that results in the formationof a covalent bond between the IRG and one of the imidazole rings foundin the histidine tag of the oligohistidine sequence, with concomitantbreak of the bond between the imidazole-reactive group and linkerfunction to yield covalently modified protein of type 3 in FIG. 1. Thecoordination bonds between metal cation M²⁺ and oligohistidine tag canbe removed by addition of a strong metal chelator such as EDTA to yielda covalently modified protein of type 4 in FIG. 1.

According to an embodiment, the fragment IRG that remains covalentlybound to the histidine tag can be covalently attached to otherfunctionalities that can be used for subsequent specific covalent ornon-covalent binding with other molecules and or other polymers.

According to an embodiment, the electron withdrawing group (abbreviated“EWG” in the figures and also known as a deactivating group) depicted inFIG. 1 and elsewhere herein may be, for example, any electronwithdrawing group, including but not limited to acyl, ester,alkylsulfonyl, arylsulfonyl, aminosulfonyl, carbamoyl, alkoxycarbonyl,nitro, and/or cyano groups, among others. The “R” group depicted in FIG.1 and elsewhere herein may be, for example, a phenyl, a substitutedalkyl, a polycyclic aromatic group, an aromatic heterocyclic group,and/or a polycyclic aromatic heterocyclic group, aryl, heteroaryl,alkyl, acyl, ester, carbamino, sulfonyl, or alkylthio, among many othertypes of functional groups. R can carry other functional groupsincluding but not limited to an azido group, alkyne group, diene group,triene group, or biotin residue, among others. The functional group mayalso be a group sufficient or capable of immobilizing the modifiedrecombinant protein to a surface. For example, the immobilizingfunctional group can be an aromatic possessing group capable of specificbinding to a surface.

According to an embodiment, the metal cations can be, for example,divalent or trivalent cations such as Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺, Co²⁺,Co³⁺, and/or Fe³⁺, among many others. The “L:” can be, for example, aligating component capable of forming coordinative bond withabovementioned metal cations including but not limited to primary,secondary, and teriary amino groups, carboxy, phosphoryl, imidazolyl,thiazolyl, oxazolyl, pyridazolyl, pyridyl, amido, sulfamido, triazolyl,and/or hydroxy groups, among many others. The linker can be, forexample, a carbon chain, or a cyclic system, or a combination thereofcapable of carrying one, two, three, four, or more ligating groups L:and covalently attached to the oxygen atom of the imidazole reactinggroup as disclosed herein, although other structures are possible.

According to another embodiment, the reaction comprises one or moreadditional optimal conditions suitable or necessary to improve and/ormaximize the chemical yield of the reaction. These conditions include,for example, use of water as the main component of solvent, and/or theuse of one or more chemical compounds capable of forming a coordinativebond with abovementioned metal cations. These additives can include, forexample, imidazole, N-methylimidazole, and/or N-alkylimidazole, amongmany others.

There is seen in FIG. 2 a schematic representation of a reaction schemefor the synthesis of a selective derivatization of an oligohistidinesequence of a recombinant protein according to another embodiment. Inthis embodiment, functional groups “A” and IRG are connected through apermanent covalent bond to produce reagents of type 5 in FIG. 2.According to this embodiment, this covalent bond is not cleaved in asubsequent stage. The initial rapid and reversible coordinative bindingof ligating groups L: through the metal cation M²⁺ to the oligohistidinetag of a recombinant produces mixed metal complex 6 in FIG. 2. The mixedmetal complex 6 serves as a template for the subsequent chemicalreaction of addition-elimination that forms a covalent bond between theIRG and one of imidazole rings of the oligohistidine tag withconcomitant elimination of leaving group EWG-O, which permanently bindsthe group to the oligohistidine tag to yield covalently modified proteinof type 7 in FIG. 2. Finally, the coordination bonds between metalcation M²⁺ and oligohistidine tag in the resultant complex can beremoved by treatment with a strong metal chelator such as EDTA to yieldthe covalently modified protein of type 8 in FIG. 2.

There is seen in FIG. 3 a schematic representation of a reaction schemefor the synthesis of a selective derivatization of an oligohistidinesequence of a recombinant protein according to another embodiment. Inthis embodiment, functional groups “A” and IRG are connected as shown instructure 9 of FIG. 3, through a carbon-oxygen bond that undergoeselimination on a subsequent step of the abovementioned process. Theinitial rapid and reversible coordinative binding of ligating groups L:through two metal cations M²⁺ to the oligohistidine tag of a recombinantprotein produces mixed metal complex 10 in FIG. 3. The mixed metalcomplex 10 serves as a template for the subsequent chemical reaction ofaddition-elimination that forms a covalent bond between the IRG and oneof imidazole rings of the oligohistidine sequence with concomitant breakof the bond between the imidazole-reactive group and linker function toyield covalently modified protein of type 11 in FIG. 3. Finally, thecoordination bonds between metal cation M²⁺ and oligohistidine tag canbe removed through can be removed by a strong metal chelator such asEDTA to yield covalently modified protein of type 12 in FIG. 3.

There is seen in FIG. 4 a schematic representation of a reaction schemefor the synthesis of a selective derivatization of an oligohistidinesequence of a recombinant protein according to another embodiment. Inthis embodiment, functional groups “A” and IRG are connected as shown instructure 9 of FIG. 3, through a carbon-oxygen bond that does notundergo elimination on a subsequent step of the abovementioned process.The initial rapid and reversible coordinative binding of ligating groupsL: through two metal cations M²⁺ to the oligohistidine tag of arecombinant protein produces mixed metal complex 14 in FIG. 4. The mixedmetal complex 14 serves as a template for the subsequent chemicalreaction of addition-elimination that form a covalent bond between theimidazole-reactive functional group IRG and one of imidazole rings ofthe oligohistidine sequence with concomitant break of the bond betweenthe imidazole-reactive group and linker function to yield covalentlymodified protein of type 11 in FIG. 4. Finally, the coordination bondsbetween metal cation M²⁺ and oligohistidine tag can be removed throughcan be removed by a strong metal chelator such as EDTA to yieldcovalently modified protein of type 12 in FIG. 3.

EXAMPLE 1

According to one embodiment is the covalent derivatization of arecombinant protein with a fluorescent group, a schematic representationof which is depicted in FIGS. 5 and 6. According to this example,recombinant protein 17 in FIG. 5 comprises an oligohistidine tag(“HHHHH”), and is derivatized with an alkynyl function through templatedalkylation with an ester 27b (see also FIG. 6 for an example of theproduction of ester 27b) followed by a “click” cycloaddition withfluorescein azide 28 to produce fluorescent protein 19. Also shown inFIG. 5 is a scan of a fluorescent image of SDS electrophoresis ofobtained samples of fluorescent protein 19 obtained by treatment withester 27b (in the presence of Cu²⁺ or Zn²⁺, or in the absence of metalcations) all after reaction with fluorescent azide 28.

According to this example, compound 21b (4-Prop-2-ynyloxy-benzaldehyde)in FIG. 6 is produced by the following procedure. A mixture of4-hydroxybenzaldehyde (6.1 g, 50 mmol), K₂CO₃ (27.6 g, 200 mmol), andpropargyl bromide (80wt %, 29.8 g, 21.6 ml, 200 mmol) in acetone washeated at reflux for 6 hours. After filtration, the filtrate wasconcentrated in vacuum. The resulting residue was dissolved in 120 mlCH₂Cl₂, washed with water, dried over Na₂SO₄, and concentrated in vacuumto yield title compounds (6.6 g, 82%) as a yellowish amorphous solid. ¹HNMR (400 MHz, CDCl₃): δ 2.20 (s, 1H), 4.69 (s, 2H), 7.10 (d, J=8.4 Hz,2H), 7.87 (d, J=8.8 Hz, 2H), 9.91 (s, 1H).

According to this example, compound 22b (Methyl2-[Hydroxy-(4-prop-2-ynyloxy-phenyl)-methyl]acrylate) in FIG. 6 isproduced by the following procedure. To a solution of aldehyde (4.8 g,30 mmol) and methyl acrylate (15 ml) in 15 ml methanol was added DABCO(3.36 g, 30 mmol) with stirring at room temperature. The mixture wasleft in darkness for 48 hours. It was then concentrated in vacuum, theresidue was dissolved in 150 ml ethyl acetate, washed with water, 1MHCl, and brine, respectively. It was then dried over Na₂SO₄, andconcentrated in vacuum. The resulting oil was purified by flashchromatography (3:1 hexane/EtOAc) to yield A-135 (2.5 g, 34%) as an oil.¹H NMR (400 MHz, CDC1₃): δ 2.52 (t, J=2.4Hz, 1H), 2.92(d, J=5.6 Hz, 1H),3.72 (s, 3H), 4.68 (d, J=2.4 Hz, 2H), 5.53 (d, J=5.2 Hz, 1H), 5.85 (s,1H), 6.33(s, 1H), 6.95 (d, J=8.8 Hz, 2H), 7.31 (d, J=8.4 Hz, 2H).

According to this example, compound 25b(Mono(1-(4-prop-2-ynyloxy-phenyl)-2-methoxycarbonyl-2-propenyl)glutaricacid N-hydroxy succinimide ester) in FIG. 6 is produced by the followingprocedure. To a solution of N-Hydroxysuccinimide (922 mg, 8 mmol) andDMAP (1466 mg, 12 mmol) in 120 ml dry THF was added glutaric anhydride 3(914 mg, 8 mmol) portionwise with stirring at 0° C. The mixture wasallowed warm to room temperature, and stirred for 4 hours andconcentrated in vacuo. The residue was dissolved in 120 ml ethylacetate, washed with 2 M HCl, brine, dried over Na₂SO₄. Afterfiltration, the filtrate was concentrated in vacuo to yield 1.8 g solidof glutaric acid N-hydroxysuccinimide monoester that was dissolved in 8ml anhydrous CH₂Cl₂ and treated with oxalyl chloride (1016 mg, 0.68 ml,8 mmol) with stifling at 0° C. The reaction mixture was allowed to warmto room temperature, to which anhydrous DMF (30 μl) was added 1 hourlater followed by stifling overnight at room temperature. The reactionmixture was concentrated to dryness in vacuum, dissolved in 6 mlanhydrous CH₂Cl₂, and added at 0° C. to a solution of alcohol 22b (1478mg, 6 mmol) and anhydrous pyridine (950 mg, 0.97 ml, 12 mmol) in 10 mlanhydrous CH₂Cl₂. The mixture was allowed to warm to room temperature,stirred for 4 hours, diluted with 120 mL 1:3hexane/EtOAc (120 mL),washed with water, 1M HCl, saturated NaHCO₃, brine, and over Na₂SO₄. Theresidue was evaporated and the residue was separated by flashchromatography (1:1 hexane/EtOAc) to yield title compound (1.3 g, 47%)as an pale yellow oil. 1H NMR (400 MHz, CDCl3): δ 2.04-2.11 (m, 2H),2.51-2.55(m, 3H), 2.68 (t, J=6.8 Hz, 2H), 2.85 (s, 4H), 3.72 (s, 3H),4.69 (d, J=2 Hz, 2H), 5.88 (s, 1H), 6.40 (s, 1H), 6.68 (S,1H), 6.96 (d,J=8.4 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ19.79, 25.57, 29.88, 32.71, 52.04, 55.79, 72.75, 75.70, 78.42, 114.80,125.64, 129.13, 130.64, 139.60, 157.63, 165.41, 168.01, 169.09, 171.16.

According to this example, compound 27b(Mono(1-(4-prop-2-ynyloxy-phenyl)-2-methoxycarbonyl-2-propenyl)glutaricacid 5S-carboxy-5-[bis(hydroxycarbonylmethyl)amino]pentylamide) in FIG.6 is produced by the following procedure. To a mixture of NTA amine (131mg, 0.5 mmol), and methanol (1 ml) was added triethylamine (101.2 mg,0.14 ml, 1 mmol) to obtain a homogeneous solution, to which a solutionof A-138B (5 ml, 0.1 M) in THF was added dropwise with stirring at 0° C.The mixture was allowed to warm to room temperature, and stirredovernight. It was then concentrated to dryness in vacuum. The residuewas purified by flash chromatography (4:1 to 1:5 CHCl₃/CH₃OH) to yieldB-071 (110 mg, 36%) as a solid. ¹H NMR (400 MHz, D2O): δ 1.21-1.70 (m,6H), 1.86(t, J=7.2 Hz, 3H), 2.18 (d, J=8.0 Hz, 2H), 2.44 (t, J=7.2 Hz,2H), 2.92 (s, 1H), 3.00-3.20 (m, 6H), 3.70 (s, 3H), 4.78 (s, 2H), 5.98(s,1H), 6.45 (s, 1H), 6.55 (s, 1H), 7.04 (d, J=8.4 Hz, 2H), 7.39 (d,J=8.4 Hz, 2H). 13C NMR (100 MHz, D2O): δ 8.21, 20.56, 23.74, 26.59,28.12, 33.04, 34.65, 38.89, 46.63, 52.45, 55.86, 68.85, 73.40, 76.78,78.56, 115.07, 127.41, 129.15, 130.39, 138.32, 157.09, 167.13, 170.52,174.04, 175.23.

According to this example, compound 28(4′,5′-bis(azidomethyl)fluoresceine) in FIG. 6 is produced by thefollowing procedure. To a mixture of 4′,5′-bis(bromomethyl)fluoresceinedibenzoate (145 mg, 0.2 mmol) and NaN₃ (65 mg, 1 mmol) was added 2 mlanhydrous DMSO and the reaction mixture was stirred at room temperaturefor 16 h. The reaction mixture was treated with NaOH (1 mL of 1M aqueoussolution) and after 1 hour acidified with excess of 1M HCl. Theresultant precipitate was collected by filtration, dried on air, andpurified by flash chromatography (25:1 CH₂Cl₂/CH₃OH) to yield titlecompound (75 mg, 85%) as a yellow-brown solid. ¹H NMR (400 MHz,DMSO-d₆): δ 4.54 (s, 4H), 6.40 (d, J=8.8 Hz, 2H), 6.47 (d, J=8.8 Hz,2H), 7.01 (d, J=7.6 Hz, 1H), 7.29 (t, J=7.6 Hz, 1H), 7.40-7.46 (m, 1H),7.51 (t, J=7.2 Hz, 1H), 7.84 (d, J=7.6 Hz, 1H), 7.92 (d, J=7.2 Hz, 1H).¹³C NMR (100 MHz, DMSO-d₆): δ 43.42, 109.60, 110.53, 112.73, 124.33,125.30, 126.71, 128.43, 128.98, 129.78, 129.87, 130.07, 133.40, 135.25,150.54, 152.29, 158.19, 170.20, 170.29.

According to this example is the sequential derivatization of protein Awith an alkynyl group followed by introduction of fluorescent groupthrough 1,3-dipolar addition. Three parallel experiments using: (a)copper(II) acetate; (b) zinc(II) acetate; and (c) blank without additionof any metal cation were all conducted using the same protocol. Asolution of ester 27b (10 μL of 0.5 mM solution in methanol) is added tomixture of Protein A (25 μL of 0.1 mM solution in water), metal salt(copper(II) acetate, or zinc acetate, or none in the blank experiment,10 μL of 0.5 mM solution in water), N-methylimidazole (12 μL of 2.5 mMsolution in methanol), methanol (50 μL), and water (50 μL). The reactionmixture was incubated for 48 h at 25° C. followed by addition ofimidazole (10 μL of 25 mM solution in methanol) to quench the reactionand prevent possible intramolecular alkylation of Protein A during thesubsequent concentration stage. Reaction mixture was evaporated usingSpeedVac system followed by addition under argon of diazide 28 (10 μL of1.5 mM solution in methanol), sodium ascorbate (10 uM of 10 mM solutionin water), and CuSO4 (10 μL of 0.5 mM solution in water) andtris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA, 4 μL or 2.5 mMsolution in methanol). The reaction mixture was incubated to 48 h at 25°C. in the darkness. Reaction mixtures were separated by SDS-PAGE using10% separating polyacrylamide gel and 4% stacking gel In-Gelfluorescence imaging was performed using Bio-Rad Gel Doc XR imagingsystem and Quantity One 1D analysis software.

Fluorescent images were obtained from the same sample at increasinglevels of fluorescence threshold. In all images the left stopcorresponds to the experiment with Cu²⁺, middle to Zn²⁺, and the rightspot to the blank experiment in the absence of metal cations.

Although the present invention has been described in connection with apreferred embodiment, it should be understood that modifications,alterations, and additions can be made to the invention withoutdeparting from the scope of the invention as defined by the claims.

What is claimed is:
 1. A method for the selective derivatization of arecombinant protein, the method comprising: contacting a recombinantprotein comprising a oligohistidine tag with a modifying compound in thepresence of a metal cation, wherein the modifying compound comprises theformulaX₁X₂X₃,wherein X₁ is an imidazole reactive group; X₂ is a linking group; and X₃is at least one ligating group; wherein said imidazole reactive groupcomprises a functional group, an electron withdrawing group, and aconnector connecting the imidazole reactive group to X₂; and furtherwherein said contacting is performed under conditions sufficient for theformation of a bond between X₃ and the oligohistidine tag utilizing saidmetal cation, and under conditions sufficient for the formation of acovalent bond between X₁ and an imidazole ring of the oligohistidine tagand concomitant separation of X₁ and X₂; and breaking the bond betweenX₃ and the oligohistidine tag using a metal chelator.
 2. The method ofclaim 1, wherein said imidazole reactive group comprises the structurerepresented by Formula (I) or (II) below:

wherein R is said functional group, R1 is said electron withdrawinggroup, and R2 is said linking group.
 3. The method of claim 1, whereinsaid electron withdrawing group is selected from the group consisting ofan acyl group, an ester group, an alkylsulfonyl group, an arylsulfonylgroup, a nitro group, a cyano group, a carbamoyl group, and combinationsthereof.
 4. The method of claim 1, wherein said functional group isselected from the group consisting of an azido group, an alkyne group,an alkene group, a diene group, a triene group, a biotin residue, aphenyl, a substituted phenyl, a polycyclic aromatic group, an aromaticheterocyclic group, a polycyclic aromatic heterocyclic group, aradioactive group, a group that possesses distinct magnetic properties,and combinations thereof.
 5. The method of claim 1, wherein X₃ comprisesa plurality of ligating groups.
 6. The method of claim 1, wherein X₃ isselected from the group consisting of a primary amino group, a secondaryamino group, a teriary amino group, a carboxy group, an imino group, aphenol group, a catechol group, a hydroxamate group, a sulfamido group,a phosphoryl group, an imidazolyl group, a thiazolyl group, an oxazolylgroup, a pyridazolyl group, a pyridyl group, a thiol group, andcombinations thereof.
 7. The method of claim 1, wherein said metalcation is selected from the group consisting of Ni²⁺, Zn²⁺, Cu²⁺, Fe²⁺,Co²⁺, Co³⁺, Fe³⁺, and combinations thereof.
 8. The method of claim 1,wherein said conditions sufficient for the formation of a bond betweenX₃ and the oligohistidine tag comprise the presence of an additive. 9.The method of claim 8, wherein the additive comprises imidazole,N-methylimidazole, or N-alkylimidazole.
 10. The method of claim 1,wherein said functional group is a flurophore.
 11. A compound for theselective derivatization of a recombinant protein comprising aoligohistidine tag, the compound comprising: the formula X₁X₂X₃, whereinX₁ is an imidazole reactive group; X₂ is a linking group; and X₃ is atleast one ligating group; wherein said imidazole reactive groupcomprises a functional group, an electron withdrawing group, and aconnector connecting the imidazole reactive group to X₂; and furtherwherein said compound is adapted to form a bond between X₃ and aoligohistidine tag of a recombinant protein in the presence of a metalcation, and further adapted to form a covalent bond between X₁ and animidazole ring of the oligohistidine tag.
 12. The compound of claim 11,wherein said imidazole reactive group comprises the structurerepresented by Formula (I) or (II) below:

wherein R is said functional group, R1 is said electron withdrawinggroup, and R2 is said linking group.
 13. The compound of claim 11,wherein X₃ comprises a plurality of ligating groups.
 14. The compound ofclaim 11, wherein said functional group is selected from the groupconsisting of an azido group, an alkyne group, a diene group, a trienegroup, a biotin residue, a phenyl, a substituted phenyl, a polycyclicaromatic group, an aromatic heterocyclic group, a polycyclic aromaticheterocyclic group, and combinations thereof.
 15. The compound of claim11, wherein X₃ is selected from the group consisting of a primary aminogroup, a secondary amino group, a teriary amino group, a carboxy group,a phosphoryl group, an imidazolyl group, a thiazolyl group, an oxazolylgroup, a pyridazolyl group, a pyridyl group, and combinations thereof.16. A method for the selective derivatization of a recombinant protein,the method comprising: contacting a recombinant protein comprising aoligohistidine tag with a modifying compound in the presence of a metalcation, wherein the modifying compound comprises the formulaX₁X₂X₃,wherein X₁ is an imidazole reactive group comprising the structurerepresented by Formula (I) or (II) below:

wherein R is a functional group, R1 is an electron withdrawing group isselected from the group consisting of an acyl group, an ester group, analkylsulfonyl group, an arylsulfonyl group, a nitro group, a cyanogroup, and combinations thereof, and R2 is a linker to a linking group;X₂ is said linking group; and X₃ is a plurality of ligating groups eachselected from the group consisting of a primary amino group, a secondaryamino group, a teriary amino group, a carboxy group, a phosphoryl group,an imidazolyl group, a thiazolyl group, an oxazolyl group, a pyridazolylgroup, a pyridyl group, and combinations thereof; and further whereinsaid contacting is performed under conditions sufficient for theformation of a bond between X₃ and the oligohistidine tag utilizing ametal cation, and under conditions sufficient for the formation of acovalent bond between X₁ and an imidazole ring of the oligohistidine tagand concomitant separation of X₁ and X₂; and breaking the bond betweenX₃ and the oligohistidine tag using a metal chelator.